Particle discrimination methods are useful in a variety of clinical assays, such as in determining the number and types of cells in a blood sample, detecting bacterial or virus particles in a body fluid sample, and assaying cell volumes and density, e.g., sperm count. Detection of non-cellular particles, such as uric acid crystals in a urine sample, is also valuable in certain clinical tests. Analysis of crystals and other particles in fluid suspension also has important industrial uses.
One method which allows rapid and efficient particle discrimination in a particle-suspension sample is flow cytometry. In this method, a suspension of particles, typically cells in a blood sample, is transported through a flow chamber where the individual particles in the sample are illuminated with one or more focused light beams. The interaction of the light beam(s) with the individual particles flowing through the chamber is detected by one or more light detectors. Commonly, the detectors are designed to measure light absorption or fluorescence emission, at specific beam or emission wavelengths, and/or light scattering at specific scattering angles. Thus each particle that passes through the flow chamber can be characterized as to one or more features related to its absorption, fluorescence, light scattering or other optical or electrical properties. The one or more properties which are measured by the detectors allow each particle to be mapped into a feature space whose axes are the light intensities or other properties which are measured by the detectors. In the ideal, the different particles in the sample map into distinct and non-overlapping regions of the feature space, allowing each particle to be analyzed based on its mapping in the feature space. Such analysis may include counting, identifying, quantifying (as to one or more physical characteristics) and/or sorting of the particles.
Two general types of light scattering measurements are routinely made in flow cytometry. Light intensity measurements made at small angles (about 1.5.degree.-13.degree. with respect to the incident light beam), usually called forward or small-angle scattering, give information on cell size (Mullaney). Forward scattering also strongly depends on the difference of refraction between cells and the extracellular medium, so that cells with a damaged membranes, for example, can be distinguished. Light intensity measurements made at an angle of about 65.degree.-115.degree. from the incident light, usually referred to as orthogonal light scattering, and these provide information about the size and degree of structuredness of particles (Visser). As an example, the inventors and co-workers have recently shown that human cytotoxic lymphocytes appear to have different structural characteristics than regulatory and B-lymphocytes, based on the greater orthogonal light scattering intensities of the cytotoxic cells (Terstappen). If the width of the illumination beam is smaller than the diameter of the particles, the pulse shape of the light at orthogonal scattered angles yields information about the length and shape of the cells.
Simultaneous light scattering measurements at different angles or in combination with absorption or fluorescence measurements have been proposed in flow cytometry methods. Simultaneous measurement of forward and orthogonal light scattering can be used to discriminate cytotoxic lymphocytes from regulatory and B-lymphocytes as indicated above (Terstappen), and lymphocytes from other peripheral leukocyte cells (Hoffman). Absorption of light in combination with light scattering is used in flow cytometry to distinguish between erythrocytes and thrombocytes, and between lymphocytes, monocytes, basophilic granulocytes, eosinophilic granulocytes, and neutrophilic granulocytes (Technicon Hemalog or Hl systems). However, this method requires staining the cells, and is therefore rather complex and may preclude using the cells for further study after cell sorting.
Light scattering measurements combined with circular dichroism (CD) and optical rotatory dispersion (ORD) also have the potential for particle discrimination in suspensions of virus particles or cells. Studies of the effect of Mie (isotropic particle) scattering on the CD and ORD spectra of DNA in viral particles (Gordon, 1972; Gordon, 1974) suggest that scattering measurements can be used to correct ORD and CD measurements in larger biological structures, such as virus particles and cells, to allow particle discrimination on the basis of characteristic ORD and CD characteristics. Differential scattering of right and left circularly polarized light, for discrimination of a number of different microorganisms, has been demonstrated (Salzman). The circular intensity differential scattering (CIDS) method is like CD, which exploits the differential absorption of left and right circularly polarized light, but takes advantage of differential scattering by helical structures, such as DNA, of right and left circularly polarized light.